The Development of a platform for HCV drug selection with a novel protease activating probe and the synthesis of NOTA derivatives as aluminium fluoride chelating agents

• Main Authors: Yen-Chen Lo, 駱彥辰
• Other Authors: Hsin-Ell Wang
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/8txgh8

碩士 === 國立陽明大學 === 生物醫學影像暨放射科學系 === 101 === Purpose: The main purpose of this study is to establish a non-invasive imaging platform related to the development of Hepatitis C pharmaceuticals and real-time monitoring. By combining Hepatitis C virus (HCV) animal model and multi-functional NS3/4A protease probes (NS3-FITC probe), this novel platform provides determination of HCV activity in vivo and may be used for anti-HCV drug selections. Method: By Lenti virus infection, we establish a stable clone cell line (HCC36-pLKO-NS3/4A) which can persistently express HCV NS3/4A protease. HCC36-pLKO-NS3/4A and the parent cell HCC36 were inoculated in Balb/C nude mice to grow tumors. Combining substrate peptide (DEDEDEDEMEEC-ASHL), Trans-Activator of Transcription peptide (TAT ,GRKKRRQRRR) and hydrophilic peptide (KKKYK, as imaging domain), we construct a novel substrate NS3 that is specific for HCV NS3/4A protease. After modified with fluorescein isothiocyanate (FITC) moiety, the cellular accumulation of NS3-FITC was determined by flow cytometry. After labeling with positron emitting radionuclide 124I at the end of NS3, 124I-NS3-FITC was characterized as a multi-modality imaging agent for both optical detection (IVIS) and in vivo nuclear imaging (SPECT and PET). We further verify the result by biopsy and bio-distribution. Result: Flow cytometry analysis showed that NS3-FITC was accumulated in HCC36-pLKO-NS3/4A cells and activated by NS3/4A protease. After 24 hr wash-out to remove un-activated NS3 probe, HCC36-pLKO-NS3/4A cells have 10 times pile-up compared to wild-type HCC36 cells. IVIS imaging indicates that there are much more NS3 probes accumulation in HCC36-pLKO-NS3/4A as well. Non-specific activation are noticed in gastrointestinal (GI) tract like stomach, small intestine (S.I.) and large intestine (L.I.). In µPET imaging, HCC36-pLKO-NS3/4A tumor region shows 4 times higher uptake in 6hr after Intravenous injection (I.V.) I124-NS3 probe. Tumor muscle ratio (T/M) is about 10 after quantification analysis. The main metabolism pathway of NS3 probe is through Urinary tract. However, as the same as IVIS imaging, non-specific activation in GI tract, such as stomach, S.I. and L.I. are seen in µPET imaging. Further verifying by biopsy and histochemistry stain, we find out the relation of FITC fluorescent signal with NS3/4A protease activity in HCC36-pLKO-NS3/4A tumor region. Conclusion: In this study, we successfully establish HCV animal model and a novel multi-functional NS3/4A probe. This platform is useful in both accelerating new drug development and in clinical diagnosis and treatment.